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The filament of a bacterial flagellum is a tube-like organelle made of single protein – flagellin, and assembled into multiple polymorphic forms. The filament can be further discretized into four subunit domains (D0, D1, D2 and D3) along the radial direction. However, it remains unclear which subunit domain plays an important role in regulating the rigidity of the filament. In this article, we address how the absence of two outer subunit domains (D2 and D3) affects the bending stiffness of the bacterium B. subtilis’ flagellar filament. We first shear off flagellar filaments from the cell body, anchor one of its ends to the wall of a microfluidic channel, and correlate the elongation of the filament with the driving background flow. A numerical model is then applied to determine the bending stiffness of the filament. We find that the bending stiffness does not change drastically when the filament transforms from normal to hyperextended forms, which is estimated to be 2-3 pN·µm2. Furthermore, B. subtilis’ flagellar filament has similar bending stiffness to Salmonella’s, though the radius of the former is almost half of that of the latter, suggesting that the rigidity comes from the inner D0 and D1 subunit domains. Ministry of Education (MOE) Submitted/Accepted version This research is supported by Ministry of Education, Singapore, under its Academic Research Funds Tier 2 MOE2018-T2-2-052.

Helicobacter pylori is a pathogenic flagellated bacterium that infects the gastroduodenal mucosa and causes peptic ulcers in humans. FliD caps the distal end of the flagellar filament and is essential in filament growth. Moreover, FliD has been studied to diagnose and prevent H. pylori infection. Here, we report structure-based molecular studies of H. pylori FliD (hpFliD). A crystal structure of hpFliD at 2.6 A resolution presents a four-domain (D2-D5) structure, where the D3 domain forms a central platform surrounded by the other three domains (D2, D4, and D5). hpFliD domains D2 and D3 structurally resemble those of FliD orthologs, whereas the D4 and D5 domains are exclusive to hpFliD. Moreover, our ELISA analysis using anti-H. pylori antibodies demonstrated that the hpFliD-specific D4 and D5 domains are highly antigenic compared to the D2 and D3 domains. Collectively, our structural and serological analyses underscore the structural role of hpFliD domains and provide a molecular basis for vaccine and diagnosis development.

In the bacterial flagellar motor, the cell-wall-anchored stator uses an electrochemical gradient across the cytoplasmic membrane to generate a turning force that is applied to the rotor connected to the flagellar filament. Existing theoretical concepts for the stator function are based on the assumption that it anchors around the rotor perimeter by binding to peptidoglycan (P). The existence of another anchoring region on the motor itself has been speculated upon, but is yet to be supported by binding studies. Due to the recent advances in electron cryotomography, evidence has emerged that polar flagellar motors contain substantial proteinaceous periplasmic structures next to the stator, without which the stator does not assemble and the motor does not function. These structures have a morphology of disks, as is the case with Vibrio spp., or a round cage, as is the case with Helicobacter pylori. It is now recognized that such additional periplasmic components are a common feature of polar flagellar motors, which sustain higher torque and greater swimming speeds compared to peritrichous bacteria such as Escherichia coli and Salmonella enterica. This review summarizes the data available on the structure, composition, and role of the periplasmic scaffold in polar bacterial flagellar motors and discusses the new paradigm for how such motors assemble and function.

The rotary bacterial flagellar motor is remarkable in biochemistry for its highly synchronized operation and amplification during switching of rotation sense. The motor is part of the flagellar basal body, a complex multi-protein assembly. Sensory and energy transduction depends on a core of six proteins that are adapted in different species to adjust torque and produce diverse switches. Motor response to chemotactic and environmental stimuli is driven by interactions of the core with small signal proteins. The initial protein interactions are propagated across a multi-subunit cytoplasmic ring to switch torque. Torque reversal triggers structural transitions in the flagellar filament to change motile behavior. Subtle variations in the core components invert or block switch operation. The mechanics of the flagellar switch have been studied with multiple approaches, from protein dynamics to single molecule and cell biophysics. The architecture, driven by recent advances in electron cryo-microscopy, is available for several species. Computational methods have correlated structure with genetic and biochemical databases. The design principles underlying the basis of switch ultra-sensitivity and its dependence on motor torque remain elusive, but tantalizing clues have emerged. This review aims to consolidate recent knowledge into a unified platform that can inspire new research strategies.

The bacterial flagellum is the principal organelle of motility in bacteria. Here, we address the question of size when applied to the chief flagellar protein flagellin and the flagellar filament. Surprisingly, nature furnishes multiple examples of 'giant flagellins' greater than a thousand amino acids in length, with large surface-exposed hypervariable domains. We review the contexts in which these giant flagellins occur, speculate as to their functions, and highlight the potential for biotechnology to build on what nature provides.

Many bacterial species swim by rotating single polar helical flagella. Depending on the direction of rotation, they can swim forward or backward and change directions to move along chemical gradients but also to navigate their obstructed natural environment in soils, sediments, or mucus. When they get stuck, they naturally try to back out, but they can also resort to a radically different flagellar mode, which we discovered here. Using high-speed microscopy, we monitored the swimming behavior of the monopolarly flagellated species Shewanella putrefaciens with fluorescently labeled flagellar filaments at an agarose-glass interface. We show that, when a cell gets stuck, the polar flagellar filament executes a polymorphic change into a spiral-like form that wraps around the cell body in a spiral-like fashion and enables the cell to escape by a screw-like backward motion. Microscopy and modeling suggest that this propagation mode is triggered by an instability of the flagellum under reversal of the rotation and the applied torque. The switch is reversible and bacteria that have escaped the trap can return to their normal swimming mode by another reversal of motor direction. The screw-type flagellar arrangement enables a unique mode of propagation and, given the large number of polarly flagellated bacteria, we expect it to be a common and widespread escape or motility mode in complex and structured environments.

Campylobacter jejunirotates a flagellum at each pole to swim through the viscous mucosa of its hosts’ gastrointestinal tracts. Despite their importance for host colonization, however, howC. jejunicoordinates rotation of these two opposing flagella is unclear. As well as their polar placement,C. jejuni’sflagella deviate from the Enterobacteriaceael norm in other ways: their flagellar motors produce much higher torque and their flagellar filament is made of two different zones of two different flagellins. To understand howC. jejuni’sopposed motors coordinate, and what contribution these factors play inC. jejunimotility, we developed strains with flagella that could be fluorescently labeled, and observed them by high-speed video microscopy. We found thatC. jejunicoordinates its dual flagella by wrapping the leading filament around the cell body during swimming in high-viscosity media and that its differentiated flagellar filament has evolved to facilitate this wrapped-mode swimming. Unexpectedly,C. jejuni’s helical body is important for facile unwrapping of the flagellar filament from the cell body during switching of swimming trajectory. Our findings demonstrate how multiple facets ofC. jejuni’sflagella and cell plan have co-evolved for optimal motility in high-viscosity environments.

Functional flagella formation is a widespread virulence factor that plays a critical role in survival and host colonization. Flagellar synthesis is a complex and highly coordinated process. The assembly of the axial structure beyond the cell membrane is mediated by export chaperone proteins that transport their cognate substrates to the export gate complex. The export chaperone FliS interacts with flagellin, the basic component used to construct the filament. Unlike enterobacteria, the gastric pathogen Helicobacter pylori produces two different flagellins, FlaA and FlaB, which exhibit distinct spatial localization patterns in the filament. Previously, we demonstrated a molecular interaction between FliS and an uncharacterized protein, HP1076, in H. pylori. Here, we present the crystal structure of FliS in complex with both the C-terminal D0 domain of FlaB and HP1076. Although this ternary complex reveals that FliS interacts with flagellin using a conserved binding mode demonstrated previously in Aquifex aeolicus, Bacillus subtilis, and Salmonella enterica serovar Typhimurium, the helical conformation of FlaB in this complex was different. Moreover, HP1076 and the D1 domain of flagellin share structural similarity and interact with the same binding interface on FliS. This observation was further validated through competitive pull-down assays and kinetic binding analyses. Interestingly, we did not observe any detrimental flagellation or motility phenotypes in an hp1076-null strain. Our localization studies suggest that HP1076 is a membrane-associated protein with a cellular localization independent of FliS. As HP1076 is uniquely expressed in H. pylori and related species, we propose that this protein may contribute to the divergence of the flagellar system, although its relationship with FliS remains incompletely elucidated.

This work aims at developing a novel method for fabricating 1D magnetite nanostructures with the help of mutagenized flagellar filaments. We constructed four different flagellin mutants displaying magnetite-binding motifs. Two of the mutants contained fragments of magnetosome-associated proteins from magnetotactic bacteria (MamI and Mms6), whereas for the other two mutants synthetic sequences were used. A magnetic selection procedure identified the mutant incorporating the loop segment from MamI to have the highest binding affinity towards magnetite. Filaments built from MamI loop-containing flagellin subunits were used as templates to form chains of magnetite nanoparticles along the filament by capturing them from suspension. Our study presents a proof of concept that flagellar filaments can be engineered to facilitite formation of 1D magnetite nanostructures under ambient conditions. In addition, it proves the interaction between MamI and magnetite, with implications for the role of the protein in magnetotactic bacteria.

Bacterial flagella are helical proteinaceous fibers, composed of the protein flagellin, that confer motility to many bacterial species. The genomes of about half of all flagellated species include more than one flagellin gene, for reasons mostly unknown. Here we show that two flagellins (FlaA and FlaB) are spatially arranged in the polar flagellum of Shewanella putrefaciens, with FlaA being more abundant close to the motor and FlaB in the remainder of the flagellar filament. Observations of swimming trajectories and numerical simulations demonstrate that this segmentation improves motility in a range of environmental conditions, compared to mutants with single-flagellin filaments. In particular, it facilitates screw-like motility, which enhances cellular spreading through obstructed environments. Similar mechanisms may apply to other bacterial species and may explain the maintenance of multiple flagellins to form the flagellar filament., It is unclear why many bacteria have more than one gene encoding flagellin, the protein that makes up flagella. Here, the authors show that a particular arrangement of two different flagellins in the polar flagellum of Shewanella putrefaciens facilitates screw-like motility through obstructed environments.

A new cryo-EM structure of the bacterial flagellar hook, which connects the motor to the flagellar filament, reveals 11 distinctive conformations of the subunit. The cooperative dynamic switching among these states offers a dramatic extension of two-state models of protein allostery.

The bacterial flagellar hook connects the helical flagellar filament to the rotary motor at its base. Bending flexibility of the hook allows the helical filaments to form a bundle behind the cell body to produce thrust for bacterial motility. The hook protein FlgE shows considerable sequence and structural similarities to the distal rod protein FlgG; however, the hook is supercoiled and flexible as a universal joint whereas the rod is straight and rigid as a drive shaft. A short FlgG specific sequence (GSS) has been postulated to confer the rigidity on the FlgG rod, and insertion of GSS at the position between Phe-42 and Ala-43 of FlgE actually made the hook straight. However, it remains unclear whether inserted GSS confers the rigidity as well. Here, we provide evidence that insertion of GSS makes the hook much more rigid. The GSS insertion inhibited flagellar bundle formation behind the cell body, thereby reducing motility. This indicates that the GSS insertion markedly reduced the bending flexibility of the hook. Therefore, we propose that the inserted GSS makes axial packing interactions of FlgE subunits much tighter in the hook to suppress axial compression and extension of the protofilaments required for bending flexibility.

Many bacteria swim through liquids or crawl on surfaces by rotating long appendages called flagella. Flagellar filaments are assembled from thousands of subunits that are exported through a narrow secretion channel and polymerize beneath a capping scaffold at the tip of the growing filament. The assembly of a flagellum uses a significant proportion of the biosynthetic capacities of the cell with each filament constituting ~1% of the total cell protein. Here, we addressed a significant question whether a flagellar filament can form a new cap and resume growth after breakage. Re-growth of broken filaments was visualized using sequential 3-color fluorescent labeling of filaments after mechanical shearing. Differential electron microscopy revealed the formation of new cap structures on broken filaments that re-grew. Flagellar filaments are therefore able to re-grow if broken by mechanical shearing forces, which are expected to occur frequently in nature. In contrast, no re-growth was observed on filaments that had been broken using ultrashort laser pulses, a technique allowing for very local damage to individual filaments. We thus conclude that assembly of a new cap at the tip of a broken filament depends on how the filament was broken.

Recent experiment of Darnton and Berg [34] showed that phase transition of bacterial flagellar filament is loading rate-dependent. The object of this paper is to describe the observed loading rate-dependent phase transition responses of the filament by using time dependent Ginzberg–Landau continuum model. We developed a finite element method (FEM) code to simulate the phase transition under a displacement-controlled loading condition (controlled helix-twist) by using viscosity-type kinetics. Our FEM simulation captures the main features of the rate-dependence: under slow loading (i.e., loading time ≫ the relaxation time) the filament phase transition is an equilibrium process and each phase grows via interface propagation on the Maxwell line; under rapid loading (i.e., loading time ≪ the relaxation time), the phase transition does not occur and the response is elastic. Our FEM model provides a tool to study the effects of loading-rate dependent phase transition for bio-filament with viscous kinetics.

The bacterial flagellar motor, one of the few rotary motors in nature, produces torque to drive the flagellar filament by ion translocation through membrane-bound stator complexes. We used the light-driven proton pump proteorhodopsin (pR) to control the proton-motive force (PMF) in vivo by illumination. pR excitation was shown to be sufficient to replace native PMF generation, and when excited in cells with intact native PMF generation systems increased motor speed beyond the physiological norm. We characterized the effects of rapid in vivo PMF changes on the flagellar motor. Transient PMF disruption events from loss of illumination caused motors to stop, with rapid recovery of their previous rotation rate after return of illumination. However, extended periods of PMF loss led to stepwise increases in rotation rate upon PMF return as stators returned to the motor. The rate constant for stator binding to a putative single binding site on the motor was calculated to be 0.06 s(-1). Using GFP-tagged MotB stator proteins, we found that transient PMF disruption leads to reversible stator diffusion away from the flagellar motor, showing that PMF presence is necessary for continued motor integrity, and calculated a stator dissociation rate of 0.038 s(-1).

Salmonella typhimurium SJW23 has a right-handed, non-helically perturbed filament of serotype gt with a unique surface pattern. Non-helical perturbations involve symmetry reduction along the five-start helical lines resulting in layer lines of fractional Bessel orders and a consequent seam. The flagellin gene, fliC 23 , which we sequenced, differs from the sequence of the canonic, plain SJW1655 flagellin, fliC 1655 . We modified discrete components of fliC 23 in order to localize, in the expressed filament, the submolecular site responsible for the non-helical perturbation. These modifications include (i) deleting the outermost domain D3 23 , (ii) replacing D3 23 with D3 1655 , (iii) substituting a hydrophilic α-helix at the interface between the neighboring domains D1 and D2 with a hydrophobic one from fliC 1655 , and (iv) substituting a serine/glycine pair in the loop connecting the modified α-helix to its neighbor; these modifications were made in the presence and absence of D3 23 . We used S. typhimurium SJW1655 both as a reference and as a source for ‘spare parts’. The symmetry of the constructs was assessed from the power spectra through changes in the layer lines at a height of 〈1/105〉 and 〈1/35〉 A − 1 , unique to the non-helical perturbation. Deleting D3 23 , either alone or in combination with various substitutions, or replacing it with D3 1655 transforms the non-helically perturbed filament into a plain one as judged by the disappearance of the typical layer lines from the power spectra. We conclude that the non-helical perturbation is a product of unique interactions in the D3 23 density shell. Whereas other minor structural changes may occur at the filaments interior, they are all helically symmetric.

Bacterial flagellar filaments can undergo a polymorphic phase transition in both vitro and vivo environments. Each bacterial flagellar filament has 12 different helical forms which are macroscopically represented by different pitch lengths and helix radii. For external mechanical force induced filament phase transitions, there is so far only one experiment performed by Hotani in 1982, who showed a very beautiful cyclic phase transition phenomenon in his experiment on isolated flagellar filaments. In the present paper, we give a detailed mechanical analysis on Hotani’s experiments. Through theoretical computations, we obtained a phase transition rule based on the phase transition mechanism. The theoretical analysis provides a foundation facilitating the establishment of phase transition theory for bacterial flagellar filaments.

Bacterial flagellum is a nano-scale motility device constructed by self-assembly. During construction of the cell-exterior filament (the ‘propeller’), subunit proteins (called flagellin) are thought to be exported through the hollow flagellum to the growing filament tip in an unfolded state. To gain insight into the unfolded state preceding any force-spectroscopy experiments on flagellin, we employed force-probe molecular dynamics simulations. Two schemes to attain an unfolded state suitable for efficient transport were examined: (i) stretching flagellin along its length; (ii) unzipping flagellin from its adjacently placed termini. Atomic-level unfolding pathways and the mechanical efforts involved under each scheme were obtained for the four-domain flagellin from S. typhimurium . Flagellin appeared stiffer and required larger unfolding forces when stretched as the relative sliding of β-strands require the breaking of multiple hydrogen bonds at once. In contrast, unzipping requires lower unfolding forces as it mainly involves unraveling β-sheets by breaking hydrogen bonds one by one.

Flagellated bacteria, such as Escherichia coli, are able to swim up gradients of chemical attractants by modulating the direction of rotation of their flagellar motors, which spin alternately clockwise (CW) and counterclockwise (CCW). Chemotactic behavior has been studied under a variety of conditions, mostly at high loads (at large motor torques). Here, we examine motor switching at low loads. Nano-gold spheres of various sizes were attached to hooks (the flexible coupling at the base of the flagellar filament) of cells lacking flagellar filaments in media containing different concentrations of the viscous agent Ficoll. The speeds and directions of rotation of the spheres were measured. Contrary to the case at high loads, motor switching rates increased appreciably with load. Both the CW-->CCW and CCW-->CW switching rates increased linearly with motor torque. Evidently, the switch senses stator-rotor interactions as well as the CheY-P concentration.

The hyperthermophilic archaeon Sulfolobus solfataricus has been shown to exhibit a complex transcriptional response to UV irradiation involving 55 genes. Among the strongest UV-induced genes was a putative pili biogenesis operon encoding a potential secretion ATPase, two pre-pilins, a putative transmembrane protein and a protein of unknown function. Electron microscopy and image reconstruction of UV-treated cells showed straight pili with 10 nm in diameter, variable in length, not bundled or polarized and composed of three evenly spaced helices, thereby clearly being distinguishable from archaeal flagella. A deletion mutant of SSO0120, the central type II/IV secretion ATPase, did not produce pili. It could be complemented by reintroducing the gene on a plasmid vector. We have named the operon ups operon for UV-inducible pili operon of Sulfolobus. Overexpression of the pre-pilins, Ups-A/B (SSO0117/0118) in Sulfolobus resulted in production of extremely long filaments. Pronounced cellular aggregation was observed and quantified upon UV treatment. This aggregation was a UV-dose-dependent, dynamic process, not inducible by other physical stressors (such as pH or temperature shift) but stimulated by chemically induced double-strand breaks in DNA. We hypothesize that pili formation and subsequent cellular aggregation enhance DNA transfer among Sulfolobus cells to provide increased repair of damaged DNA via homologous recombination.

The flagellar filament of Salmonella is an assembly of a single protein, flagellin. There are different helical and straight shape filaments from wild type and mutants. Raman and Raman optical activity (ROA) spectroscopies were employed to characterize the structure of flagellar filaments. The Raman spectra of normal and two straight filaments were almost same, but in the ROA spectra, intensive ROA peaks at amide I, amide III and skeletal vibrational regions were observed from only one of two straight filaments, L-type straight filaments. When the filaments were depolymerized or fragmented, the ROA bands disappeared on the scale of the large ROA intensities. It is thought that the ROA bands of L-type flagellar filaments are related to their shape, length and possible higher level chiral organization.

The bacterial flagellar filament controls the swimming mode of bacteria by its polymorphic structural change. We investigated dynamics of the flagellar filament by massive molecular dynamic simulation and found that three types of inter-molecular interactions, permanent, sliding and switch interactions, play essential roles in the structural change. The polymorphic structure of the flagellar filament is thought to be due to energy frustration between intra- and inter-molecular interactions.

The bacterial flagellar filament is a helical propeller for the rapid swimming of bacteria. Its gently curved and twisted tubular structure is made of a single protein, flagellin, by its self-assembly in a helical manner. The mechanism for the formation of the left-handed helical tube for swimming and its dynamic polymorphic transition into right-handed forms for tumbling in response to quick reversal of the motor rotation has been revealed in the filament and protofilament structures at atomic detail obtained by convergent application of electron cryomicroscopy, X-ray fiber diffraction and X-ray crystallography with new technological devices in each of these methods.